Integrins are heterodimeric cell surface glycoproteins that are composed of non-covalently associated .alpha. and .beta. subunits. Sixteen .alpha. subunits and eight .beta. subunits have been identified. Over 20 different combinations of these subunits have been found.
Integrins anchor cells to their surroundings by mediating cell-matrix and cell--cell interactions (for review, see Hemler, M. E. Annu. Rev. Immunol. 8:365-400 (1990); and Hynes, R. O. Cell 69:11-25 (1992) and citations therein). Recognition of the arginine-glycine-aspartic acid (RGD) sequence in extracellular matrix proteins by the integrins is a fundamental phenomenon in cell-matrix interaction. Fibronectin is the prototype of a RGD-containing protein. In addition, a large number of other matrix molecules in mammals, birds, frogs, and insects mediate cell adhesion via their RGD-sequence. Integrin heterodimers containing a .beta.1, .beta.3, .beta.5, or .beta.6 subunit can form RGD-dependent receptors (Ruoslahti, E., J. Clin. Invest. 87:1-5 (1991); Busk, M. et al., J. Biol. Chem. 267:7875-7881 (1992); and Elices, M. J. et al., J. Cell Biol. 112:169-181 (1991)). In the .beta.1 subunit the RGD-binding site has been mapped to the amino terminal half of the molecule and there is some evidence suggestive of the possibility that the a subunit may influence this interaction (Shih, D. T. et al., J. Cell Biol. 122:1361-1371 (1993)). All together, ten integrin heterodimers share the common .beta.1 subunit, and therefore have also the putative RGD-binding site. Most of the .beta.1 integrins have, however, additional mechanisms for ligand binding. Denatured fibrillar collagens are recognized by the RGD-dependent integrins, like .alpha.5.beta.1, whereas native collagens interact with integrins in a RGD-independent manner (Gullberg, D. et al., EMBO J. 11:3865-3873 (1992)).
Two integrins, the .alpha.1.beta.1 and .alpha.2.beta.1 heterodimers, are the major cellular receptors for native collagens and like all integrins their interaction with ligands is dependent on divalent cations (Staatz, W. D. et al., J. Biol. Chem. 266:7363-7387 (1991)). Integrin .alpha.2.beta.1 is expressed for example on epithelial cells, platelets, granulation tissue cells, and various cancer cells. Biological phenomena in which .alpha.2.beta.1 integrin activity (function) is essential include collagen-induced platelet aggregation, cell migration on collagen, and cell-dependent reorganization of collagen fibers. In cancer biology, the .alpha.2.beta.1 integrin has been associated with an invasive cell phenotype and it can be a marker for aggressive melanoma. On the other hand, overexpression of .alpha.2.beta.1 integrin in breast cancer cells restores the normal phenotype. Like other integrins, .alpha.2.beta.1 can also generate signals regulating cellular functions and gene expression. Especially, the MRNA levels of collagenase-1 seem to be controlled by .alpha.2.beta.1 integrin.
The .alpha.1 and .alpha.2 subunits differ in their structure from all other .beta.1 associated .alpha. subunits in the sense that they contain a special "inserted" domain, the I domain, which resembles the A domain found e.g. in von Willenbrand factor (Michishita, M. et al., Cell 72:857-867 (1993)). It is evident that .alpha.1I and .alpha.2I domains are responsible for the primary recognition of collagen by the corresponding integrins (Kamata, T. et al., J. Biol. Chem. 269:9659-9663 (1994); Kamata, T. et al., J. Biol. Chem. 269:26006-26101 (1994); Kern, A. et al., J. Biol. Chem. 269:22811-22816 (1994)). Two other ligands for .alpha.2.beta.1 integrin, namely lamin-1 and echovirus-1, both bind to the .alpha.2I-domain, as well. However, echovirus-1 seems to recognize a different site in .alpha.2I domain than the matrix proteins (Bergelson, J. M. et al., J. Clin. Invest. 92:232-239 (1993)).
The binding sites of .alpha.1.beta.1 and .alpha.2.beta.1 integrins in collagens have been localized in the triple-helical areas of the molecules (Eble, J. A. et al., EMBO J. 12:4795-4802 (1993); Gulberg, D. et al., EMBO J. 11:3865-3873 (1992)). One peptide sequence derived from the collagen a chain has been reported to block integrin-collagen interaction, but in many studies it has been ineffective and it probably does not represent the actual binding site in collagen (Cardarelli, P. M. et al., J. Biol. Chem. 267:23159-23164 (1992); Pfaff, M. et al., Exp. Cell Res. 206:167-176 (1993); and Tuckwell, D. et al., J. Cell Sci. 108:1629-1637 (1995)). More likely collagen-receptor integrins recognize amino acid residues from more than one collagen .alpha. chain. In type IV collagen-.alpha.1.beta.1 integrin interaction, the importance of one arginine and two aspartic acid residues, all from different .alpha. chains of the collagen, has been indicated (Eble, J. A. et al., EMBO J. 12:4795-4802 (1993)).
The known matrix molecule ligands for .alpha.2.beta.1 integrin do not contain an RKK(H) sequence. An arginine-rich linear peptide comprising an RKK sequence, derived from a human immunodeficiency virus Tat protein has been shown to interact with .alpha.V.beta.5 integrin (Vogel, B. E. et al., J. Cell Biol. 121:461-468 (1993)). However the integrin-peptide interaction was found to be stable in the presence of EDTA, indicating a distinct binding mechanism. There is also a previously described heparin sulfate binding sequence in fibronectin containing an RKK sequence motif, which, however, obviously is non-functional in terms of the integrin r.alpha.2I domain binding (Drake et al., J. Biol. Chem. 268:15859-15867 (1993)).
The venom from several snake species contains disintegrin-like proteins, which block platelet integrin function and are responsible for the anticoagulation effect of the venoms. These proteins have helped to understand the molecular mechanisms of integrin function and they have potential value also in the development of new drugs. Many of the disintegrins have an RGD-sequence and they inhibit the function of platelet .alpha.IIb.beta.3 and .alpha.V.beta.3 integrins. In jararhagin, a disintegrini/metalloproteinase from pit viper Bothrops jararaca, the sequence ECD replaces RGD (Paine, M. J. I. et al., J. Biol. Chem. 267:22869-22876 (1992)). Jararhagin is a potent inhibitor of collagen-induced platelet aggregation and its effect is based on the inhibition of .alpha.2.beta.1 integrin function (De Luca, M. et al., Biochem. Biophys. Res. Commun. 206:570-576 (1995)). The exact mechanism of its action has been unknown. Integrin .alpha.2.beta.1 may also interact with jaracetin, a snake venom protein containing the disintegrin domain of jararhagin, but the interaction seems to be weaker than with jararhagin (De Luca, M. et al., Biochem. Biophys. Res. Commun. 206:570-576 (1995)).